The long-term objective of this project is to understand how to modulate tight intercellular junctions for improving drug delivery by regulating protein interactions that mediate the intercellular junctions. The short- term objectives of this proposal are to understand the mechanisms of E- cadherin-mediated intercellular junctions and to evaluate the ability of cadherin peptides to modulate cadherin interactions for improving paracellular drug delivery. Tight intercellular junctions are mediated, at least in part, by cell surface proteins called E-cadherins. Cadherin- mediated cell-cell adhesion is produced by homophilic interactions in which E-cadherin molecules from one cell interact with E-cadherin molecules from another cell. Our hypothesis is that peptide sequences similar to those found in the binding region of cadherin-cadherin interactions can be used to modulate E-cadherin-mediated cell adhesion in an equilibrium fashion; thus, they can be used to identify the mechanisms of intercellular junction formation by E-cadherins. Furthermore, peptides derived from E-cadherin sequence may modulate cadherin-cadherin interactions and create channels for paracellular drug delivery. These proposed studies would allow us to understand the mechanisms of modulation of biological barriers such as the intestinal mucosa and the blood-brain barrier (BBB). For this purpose, monolayers of bovine-brain microvessel endothelial cells (BBMECs), colon adenocarcinoma-2 (Caco-2) cells, and Madin-Darby Canine Kidney (MDCK) cells will be used as in vitro models for the BBB, intestinal mucosa, and kidney barriers; these cells have E-cadherins in their tight intercellular junctions. Therefore, this work will be focused on comparing the ability and selectivity of cadherin peptides to regulate the E-cadherin mediated cell-cell adhesion in these different types of cells. Secondly, the extracellular domain (EC domain) proteins from E- cadherin will be overexpressed to elucidate the homophilic interaction of E-cadherins; this study will also involve: a) equilibrium binding studies between cadherin peptides and EC-domain proteins, b) structural studies of the EC-domain proteins, and c) determination of the bound conformational of cadherin peptides to EC-domain protein. The peptide/protein structures will be determined by NMR, CD, and molecular modeling. Finally, cadherin peptides will be optimized and used to modulate intercellular junctions for improving paracellular delivery of marker molecules using in vitro cell culture models. Understanding the mechanisms of E-cadherin-E-cadherin interactions may be essential for understanding the integrity of biological barriers in general and the role of E-cadherins in maintaining cell-cell adhesion in different tissues, including tumors.